Solid dressing for treating wounded tissue

ABSTRACT

Disclosed are solid dressings for treated wounded tissue in mammalian patients, such as a human, comprising a haemostatic layer consisting essentially of fibrinogen and a fibrinogen activator, wherein the fibrinogen is present in an amount between 3.0 mg/cm 2  of the wound facing surface of the dressing and 13.0 mg/cm 2  of the wound facing surface of the dressing. Also disclosed are methods for treating wounded tissue.

The present application is a continuation of U.S. patent applicationSer. No. 11/882,876, entitled, “Solid Dressing for Treating WoundedTissue,” filed Aug. 6, 2007, which is incorporated herein by reference.

FIELD OF THE INVENTION Background of the Invention

The present invention relates to a solid dressing for treating woundedtissue in a mammalian patient, such as a human. The materials andmethods available to stop bleeding in pre-hospital care (gauzedressings, direct pressure, and tourniquets) have, unfortunately, notchanged significantly in the past 2000 years. See L. Zimmerman et al.,Great Ideas in the History of Surgery (San Francisco, Calif.: NormanPublishing; 1993), 31. Even in trained hands they are not uniformlyeffective, and the occurrence of excessive bleeding or fatal hemorrhagefrom an accessible site is not uncommon. See J. M. Rocko et al., J.Trauma 22:635 (1982).

Mortality data from Vietnam indicates that 10% of combat deaths were dueto uncontrolled extremity hemorrhage. See SAS/STAT Users Guide, 4th ed.(Cary, N.C.: SAS Institute Inc.; 1990). Up to one third of the deathsfrom ex-sanguination during the Vietnam War could have been prevented bythe use of effective field hemorrhage control methods. See SAS/STATUsers Guide, 4th ed. (Cary, N.C.: SAS Institute Inc.; 1990).

Although civilian trauma mortality statistics do not provide exactnumbers for pre-hospital deaths from extremity hemorrhage, case andanecdotal reports indicate similar occurrences. See J. M. Rocko et al.These data suggest that a substantial increase in survival can beaffected by the pre-hospital use of a simple and effective method ofhemorrhage control.

There are now in use a number of newer haemostatic agents that have beendeveloped to overcome the deficiencies of traditional gauze bandages.These haemostatic agents include the following:

-   -   Microporous polysaccharide particles (TraumaDEX®, Medafor Inc.,        Minneapolis, Minn.);    -   Zeolite (QuikClot®, Z-Medica Corp, Wallington, Conn.);    -   Acetylated poly-N-acetyl glucosamine (Rapid Deployment Hemostat™        (RDH), Marine Polymer Technologies, Danvers, Mass.);    -   Chitosan (HemCon® bandage, HemCon Medical Technologies Inc.,        Portland Oreg.);    -   Liquid Fibrin Sealants (Tisseel VH, Baxter, Deerfield, Ill.)    -   Human fibrinogen and thrombin on equine collagen (TachoComb-S,        Hafslund Nycomed Pharma, Linz, Austria);    -   Microdispersed oxidized cellulose (m•doc™, Alltracel Group,        Dublin, Ireland);    -   Propyl gallate (Hemostatin™, Analytical Control Systems Inc.,        Fishers, Ind.);    -   Epsilon aminocaproic acid and thrombin (Hemarrest™ patch,        Clarion Pharmaceuticals, Inc.);    -   Purified bovine corium collagen (Avitene® sheets (non-woven web        or Avitene Microfibrillar Collagen Hemostat (MCH), Davol, Inc.,        Cranston, R.I.);    -   Controlled oxidation of regenerated cellulose (Surgicel®,        Ethicon Inc., Somerville, N.J.);    -   Aluminum sulfate with an ethyl cellulose coating (Sorbastace        Microcaps, Hemostace, LLC, New Orleans, La.);    -   Microporous hydrogel-forming polyacrylamide (BioHemostat,        Hemodyne, Inc., Richmond Va.); and    -   Recombinant activated factor VII (NovoSeven®, NovoNordisk Inc.,        Princeton, N.J.).        These agents have met with varying degrees of success when used        in animal models of traumatic injuries and/or in the field.

One such agent is a starch-based haemostatic agent sold under the tradename TraumaDEX™. This product comprises macroporous polysaccharideparticles that are poured directly into or onto a wound. The particlesappear to exert their haemostatic effect by absorbing water from theblood and plasma in the wound, resulting in the accumulation andconcentration of clotting factors and platelets. In two studies of alethal groin wound model, however, this agent showed no meaningfulbenefit over standard gauze dressings. See McManus et al., BusinessBriefing: Emergency Medical Review 2005, pp. 76-79 (presently availableon-line at www.touchbriefings.com/pdf/1334/Wedmore.pdf).

Another particle-based agent is QuickClot™ powder, a zeolite granularhaemostatic agent that is poured directly into or onto a wound. Thezeolite particles also appear to exert their haemostatic effect throughfluid absorption, which cause the accumulation and concentration ofclotting factors and platelets. Although this agent has been usedsuccessfully in some animal studies, there remains concern about theexothermic process of fluid absorption by the particles. Some studieshave shown this reaction to produce temperatures in excess of 143° C. invitro and in excess of 50° C. in vivo, which is severe enough to causethird-degree burns. See McManus et al., Business Briefing: EmergencyMedical Review 2005, at 77. The exothermic reaction of QuikClot™ hasalso been observed to result in gross and histological tissue changes ofunknown clinical significance. Acheson et al., Trauma 59:865-874 (2005).

Unlike these particle-based agents, the Rapid Deployment Hemostat™appears to exert its haemostatic effect through red blood cellaggregation, platelet activation, clotting cascade activation and localvasoconstriction. The Rapid Deployment Hemostat™ is an algae-deriveddressing composed of poly-N-acetyl-glucosamine. While the originaldressing design was effective in reducing minor bleeding, it wasnecessary to add gauze backing in order to reduce blood loss in swinemodels of aortic and liver injury. See McManus et al., BusinessBriefing: Emergency Medical Review 2005, at 78.

Another poly-N-acetyl-glucosamine-derived dressing is the HemCon™Chitosan Bandage, which is a freeze-dried chitosan dressing purportedlydesigned to optimize the mucoadhesive surface density and structuralintegrity of the chitosan at the site of the wound. The HemCon™ ChitosanBandage apparently exerts its haemostatic effects primarily throughadhesion to the wound, although there is evidence suggesting it may alsoenhance platelet function and incorporate red blood cells into the clotit forms on the wound. This bandage has shown improved hemostasis andreduced blood loss in several animal models of arterial hemorrhage, buta marked variability was observed between bandages, including thefailure of some due to inadequate adherence to the wound. See McManus etal., Business Briefing: Emergency Medical Review 2005, at 79.

Liquid fibrin sealants, such as Tisseel VH, have been used for years asan operating room adjunct for hemorrhage control. See J. L. Garza etal., J. Trauma 30:512-513 (1990); H. B. Kram et al., J. Trauma30:97-101(1990); M. G. Ochsner et al., J. Trauma 30:884-887 (1990); T.L. Matthew et al., Ann. Thorac. Surg. 50:40-44 (1990); H. Jakob et al.,J. Vasc. Surg., 1:171-180 (1984). The first mention of tissue glue usedfor hemostasis dates back to 1909. See Current Trends in Surgical TissueAdhesives: Proceedings of the First International Symposium on SurgicalAdhesives, M. J. MacPhee et al., eds. (Lancaster, Pa.: TechnomicPublishing Co; 1995). Liquid fibrin sealants are typically composed offibrinogen and thrombin, but may also contain Factor XIII/XIIIa, eitheras a by-product of fibrinogen purification or as an added ingredient (incertain applications, it is therefore not necessary that FactorXIII/Factor XIIIa be present in the fibrin sealant because there issufficient Factor XIII/XIIIa, or other transaminase, endogenouslypresent to induce fibrin formation). As liquids, however, these fibrinsealants have not proved useful for treating traumatic injuries in thefield.

Dry fibrinogen-thrombin dressings having a collagen support (e.g.TachoComb™, TachoComb™ H and TachoSil available from Hafslund NycomedPharma, Linz, Austria) are also available for operating room use in manyEuropean countries. See U. Schiele et al., Clin. Materials 9:169-177(1992). While these fibrinogen-thrombin dressings do not require thepre-mixing needed by liquid fibrin sealants, their utility for fieldapplications is limited by a requirement for storage at 4° C. and thenecessity for pre-wetting with saline solution prior to application tothe wound. These dressings are also not effective against high pressure,high volume bleeding. See Sondeen et al., J. Trauma 54:280-285 (2003).

A dry fibrinogen/thrombin dressing for treating wounded tissue is alsoavailable from the American Red Cross (ARC). As disclosed in U.S. Pat.No. 6,762,336, this particular dressing is composed of a backingmaterial and a plurality of layers, the outer two of which containfibrinogen (but no thrombin) while the inner layer contains thrombin andcalcium chloride (but no fibrinogen). While this dressing has showngreat success in several animal models of hemorrhage, the bandage isfragile, inflexible, and has a tendency to break apart when handled. SeeMcManus et al., Business Briefing: Emergency Medical Review 2005, at 78;Kheirabadi et al., J. Trauma 59:25-35 (2005). In addition, U.S. Pat. No.6,762,336 teaches that this bandage should contain 15 mg/cm² offibrinogen to successfully pass a porcine arteriotomy test that is lessrobust than that disclosed in this application (see Example XI).Moreover, although U.S. Pat. No. 6,762,336 discloses that bandagescomprising two layers of fibrinogen, each with a concentration of 4mg/cm² to 15 mg/cm² may provide effective control of hemorrhage, itfurther teaches that “fibrinogen dose is related to quality. The higherdose is associated with more firm and tightly adhered clots. While lowerfibrinogen doses are effective for hemorrhage control during the initial60 minutes, longer term survival will likely depend on clot quality.”

Other fibrinogen/thrombin-based dressings have also been proposed. Forexample, U.S. Pat. No. 4,683,142 discloses a resorptive sheet materialfor closing and healing wounds which consists of a glycoprotein matrix,such as collagen, containing coagulation proteins, such as fibrinogenand thrombin. U.S. Pat. No. 5,702,715 discloses a reinforced biologicalsealant composed of separate layers of fibrinogen and thrombin, at leastone of which also contains a reinforcement filler such as PEG, PVP, BSA,mannitol, FICOLL, dextran, myo-inositol or sodium chlorate. U.S. Pat.No. 6,056,970 discloses dressings composed of a bioabsorbable polymer,such as hyaluronic acid or carboxymethylcellulose, and a haemostaticcomposition composed of powdered thrombin and/or powdered fibrinogen.U.S. Pat. No. 7,189,410 discloses a bandage composed of a backingmaterial having thereon: (i) particles of fibrinogen; (ii) particles ofthrombin; and (iii) calcium chloride. U.S. Patent ApplicationPublication No. US 2006/0155234 A1 discloses a dressing composed of abacking material and a plurality of fibrinogen layers which havediscrete areas of thrombin between them. To date, none of thesedressings have been approved for use or are available commercially.

Accordingly, there remains a need in the art for a solid dressing thatcan be used to treat wounded tissue, particularly wounded tissueresulting from traumatic injury in the field.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a soliddressing that can treat wounded mammalian tissue, particularly woundedtissue resulting from a traumatic injury. It is further an object of thepresent invention to provide a method of treating wounded mammaliantissue, particularly human tissue. Other objects, features andadvantages of the present invention will be set forth in the detaileddescription of preferred embodiments that follows, and will in part beapparent from that description and/or may be learned by practice of thepresent invention. These objects and advantages will be realized andattained by the compositions and methods described in this specificationand particularly pointed out in the claims that follow.

In accordance with these and other objects, a first embodiment of thepresent invention is direct to a solid dressing for treating woundedtissue in a mammal comprising at least one haemostatic layer consistingessentially of fibrinogen and a fibrinogen activator, wherein thefibrinogen is present in an amount between about 3.0 mg/cm² of thesurface area of the wound facing side of the dressing and 13.0 mg/cm² ofthe surface area of the wound facing side of the dressing.

Another embodiment is directed to a method of treating wounded tissueusing a solid dressing comprising at least one haemostatic layerconsisting essentially of fibrinogen and a fibrinogen activator, whereinthe fibrinogen is present in an amount between about 11.0 mg/cm² of thesurface area of the wound facing side of the dressing and 13.0 mg/cm² ofthe surface area of the wound facing side of the dressing.

It is to be understood that the foregoing general description and thefollowing detailed description of preferred embodiments are exemplaryand explanatory only and are intended to provide further explanation,but not limitation, of the invention as claimed herein.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1C are graphs showing the results achieved in Example 1.

FIG. 2 is a diagram of the set-up for the ex vivo porcine arteriotomoyassay described herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsmentioned herein are incorporated by reference.

As used herein, use of a singular article such as “a,” “an,” and “the”is not intended to excluded pluralities of the article's object unlessthe context clearly and unambiguously dictates otherwise.

“Patient” as used herein refers to human or animal individuals in needof medical care and/or treatment.

“Wound” as used herein refers to any damage to any tissue of a patientwhich results in the loss of blood from the circulatory system and/orany other fluid from the patient's body. The tissue may be an internaltissue, such as an organ or blood vessel, or an external tissue, such asthe skin. The loss of blood may be internal, such as from a rupturedorgan, or external, such as from a laceration. A wound may be in a softtissue, such as an organ, or in hard tissue, such as bone. The damagemay have been caused by any agent or source, including traumatic injury,infection or surgical intervention.

“Resorbable material” as used herein refers to a material that is brokendown spontaneously and/or by the mammalian body into components whichare consumed or eliminated in such a manner as not to interferesignificantly with wound healing and/or tissue regeneration, and withoutcausing any significant metabolic disturbance.

“Stability” as used herein refers to the retention of thosecharacteristics of a material that determine activity and/or function.

“Suitable” as used herein is intended to mean that a material does notadversely affect the stability of the dressings or any componentthereof.

“Binding agent” as used herein refers to a compound or mixture ofcompounds that improves the adherence and/or cohesion of the componentsof the haemostatic layer(s) of the dressings.

“Solubilizing agent” as used herein refers to a compound or mixture ofcompounds that improves the dissolution of a protein or proteins inaqueous solvent.

“Filler” as used herein refers to a compound or mixture of compoundsthat provide bulk and/or porosity to the haemostatic layer(s) of adressing.

“Release agent” as used herein refers to a compound or mixture ofcompounds that facilitates removal of a dressing from a manufacturingmold.

“Foaming agent” as used herein refers to a compound or mixture ofcompounds that produces gas when hydrated under suitable conditions.

“Solid” as used herein is intended to mean that the dressing will notsubstantially change in shape or form when placed on a rigid surface,wound-facing side down, and then left to stand at room temperature for24 hours.

A first preferred embodiment of the present invention is directed to asolid dressing for treating wounded tissue in a patient which comprisesa haemostatic layer consisting of fibrinogen and a fibrinogen activator,wherein the fibrinogen is present in an amount between 3.0 mg/cm² of thesurface area of the wound facing side of the dressing and 13.0 mg/cm² ofthe surface area of the wound facing side of the dressing, all valuesbeing ±0.09 mg/cm².

As used herein, “consisting essentially of” is intended to mean that thefibrinogen and the fibrinogen activator are the only necessary andessential ingredients of the haemostatic layer(s) of the solid dressingwhen it is used as intended to treat wounded tissue. Accordingly, thehaemostatic layer may contain other ingredients in addition to thefibrinogen and the fibrinogen activator as desired for a particularapplication, but these other ingredients are not required for the soliddressing to function as intended under normal conditions, i.e. theseother ingredients are not necessary for the fibrinogen and fibrinogenactivator to react and form enough fibrin to reduce the flow of bloodand/or fluid from normal wounded tissue when that dressing is applied tothat tissue under the intended conditions of use. If, however, theconditions of use in a particular situation are not normal, for examplethe patient is a hemophiliac suffering from Factor XIII deficiency, thenthe appropriate additional components, such as Factor XIII/XIIIa or someother transaminase, may be added to the haemostatic layer(s) withoutdeviating from the spirit of the present invention. Similarly, the soliddressing of the present invention may contain one or more of thesehaemostatic layers as well as one or more other layers, such as one ormore support layers (e.g. a backing material or an internal supportmaterial) and release layers.

Other preferred embodiments of the present invention include similarsolid dressings wherein the fibrinogen is present in an amount between11.0 mg/cm² of the surface area of the wound facing side of the dressingand 13.0 mg/cm² of the surface area of the wound facing side of thedressing, all values being ±0.09 mg/cm². Other preferred embodimentsinclude similar solid dressings wherein the fibrinogen is present in anamount between 3.0 mg/cm² and 9.0 mg/cm² Still other preferredembodiments are directed to similar solid dressings wherein the amountof fibrinogen is: 3.0 mg/cm² of the surface area of the wound facingside of the dressing; 5.0 mg/cm²; 7.0 mg/cm²; 9.0 mg/cm²; 11.0 mg/cm²;or 13.0 mg/cm² (all values being ±0.09 mg/cm²).

Another preferred embodiment of the present invention is directed to amethod for treating wounded tissue in a mammal, comprising placing asolid dressing of the present invention to wounded tissue and applyingsufficient pressure to the dressing for a sufficient time for enoughfibrin to form to reduce the loss of blood and/or other fluid from thewound.

According to certain embodiments of the present invention, thehaemostatic layer(s) of the solid dressing is formed or cast as a singlepiece. According to certain other embodiments of the present invention,the haemostatic layer is made or formed into or from a single source,e.g. an aqueous solution containing a mixture of the fibrinogen and thefibrinogen activator. With each of these embodiments of the presentinvention, the haemostatic layer(s) is preferably substantiallyhomogeneous throughout.

According to other preferred embodiments, the haemostatic layer(s) ofthe solid dressing are composed of a plurality of particles, each ofwhich consists essentially of fibrinogen and a fibrinogen activator.According to such embodiments, the haemostatic layer may also contain abinding agent to facilitate or improve the adherence of the particles toone another and/or to any support layer(s). Illustrative examples ofsuitable binding agents include, but are not limited to, sucrose,mannitol, sorbitol, gelatin, hyaluron and its derivatives, such ashyaluronic acid, maltose, povidone, starch, chitosan and itsderivatives, and cellulose derivatives, such as carboxymethylcellulose,as well as mixtures of two or more thereof.

The haemostatic layer(s) of the solid dressing may also optionallycontain one or more suitable fillers, such as sucrose, lactose, maltose,silk, fibrin, collagen, albumin, hyaluron and its derivatives, such ashyaluronic acid, polysorbate (Tween™), chitin, chitosan and itsderivatives, such as NOCC-chitosan, alginic acid and salts thereof,cellulose and derivatives thereof, proteoglycans, glycolic acidpolymers, lactic acid polymers, glycolic acid/lactic acid co-polymers,and mixtures of two or more thereof.

The haemostatic layer of the solid dressing may also optionally containone or more suitable solubilizing agents, such as sucrose, dextrose,mannose, trehalose, mannitol, sorbitol, albumin, hyaluron and itsderivatives, such as hyaluronic acid, sorbate, polysorbate (Tween™),sorbitan (SPAN™) and mixtures of two or more thereof.

The haemostatic layer of the solid dressing may also optionally containone or more suitable foaming agents, such as a mixture of aphysiologically acceptable acid (e.g. citric acid or acetic acid) and aphysiologically suitable base (e.g. sodium bicarbonate or calciumcarbonate). Other suitable foaming agents include, but are not limitedto, dry particles containing pressurized gas, such as sugar particlescontaining carbon dioxide (see, e.g., U.S. Pat. No. 3,012,893) or otherphysiologically acceptable gases (e.g. Nitrogen or Argon), andpharmacologically acceptable peroxides.

The haemostatic layer(s) of the solid dressing may also optionallycontain a suitable source of calcium ions, such as calcium chloride,and/or a fibrin cross-linker, such as a transaminase (e.g. FactorXIII/XIIIa) or glutaraldehyde.

The haemostatic layer of the solid dressing is preferably prepared bymixing aqueous solutions of the fibrinogen and the fibrinogen activatorunder conditions which minimize the activation of the fibrinogen by thefibrinogen activator. The mixture of aqueous solutions is then subjectedto a process such as lyophilization or free-drying to reduce themoisture content to the desired level, i.e. to a level where thedressing is solid and therefore will not substantially change in shapeor form upon standing, wound-facing surface down, at room temperaturefor 24 hours. Similar processes that achieve the same result, such asdrying, spray-drying, vacuum drying and vitrification, may also beemployed.

As used herein, “moisture content” refers to the amount freely-availablewater in the dressing. “Freely-available” means the water is not boundto or complexed with one or more of the non-liquid components of thedressing. The moisture content referenced herein refers to levelsdetermined by procedures substantially similar to the FDA-approved,modified Karl Fischer method (Meyer and Boyd, Analytical Chem.,31:215-219, 1959; May et al., J. Biol. Standardization, 10:249-259,1982; Centers for Biologics Evaluation and Research, FDA, Docket No.89D-0140, 83-93; 1990) or by near infrared spectroscopy. Suitablemoisture content(s) for a particular solid dressing may be determinedempirically by one skilled in the art depending upon the intendedapplication(s) thereof.

For example, in certain embodiments of the present invention, highermoisture contents are associated with more flexible solid dressings.Thus, in solid dressings intended for extremity wounds, it may bepreferred to have a moisture content of at least 6% and even morepreferably in the range of 6% to 44%.

Similarly, in other embodiments of the present invention, lower moisturecontents are associated with more rigid solid dressings. Thus, in soliddressings intended for flat wounds, such as wounds to the abdomen orchest, it may be preferred to have a moisture content of less than 6%and even more preferably in the range of 1% to 6%.

Accordingly, illustrative examples of suitable moisture contents forsolid dressings include, but are not limited to, the following (eachvalue being ±0.9%): less than 53%; less than 44%; less than 28%; lessthan 24%; less than 16%; less than 12%; less than 6%; less than 5%; lessthan 4%; less than 3%; less than 2.5%; less than 2%; less than 1.4%;between 0 and 12%, non-inclusive; between 0 and 6%; between 0 and 4%;between 0 and 3%; between 0 and 2%; between 0 and 1%; between 1 and 16%;between 1 and 11%; between 1 and 8%; between 1 and 6%; between 1 and 4%;between 1 and 3%; between 1 and 2%; and between 2 and 4%.

The fibrinogen in the haemostatic layer(s) of the solid dressings may beany suitable fibrinogen known and available to those skilled in the art.A specific fibrinogen for a particular application may be selectedempirically by one skilled in the art. As used herein, the term“fibrinogen” is intended to include mixtures of fibrinogen and smallamounts of Factor XIII/Factor XIIIa, or some other such transaminaseSuch small amounts are generally recognized by those skilled in the artas usually being found in mammalian fibrinogen after it has beenpurified according to the methods and techniques presently known andavailable in the art, and typically range from 0.1 to 20 Units/mL.

Preferably, the fibrinogen employed as the fibrinogen component of thesolid dressing is a purified fibrinogen suitable for introduction into amammal Typically, such fibrinogen is a part of a mixture of human plasmaproteins which include Factor XIII/XIIIa and have been purified to anappropriate level and virally inactivated. A preferred aqueous solutionof fibrinogen for preparation of a solid dressing contains around 37.5mg/mL fibrinogen at a pH of around 7.4±0.1. Suitable fibrinogen for useas the fibrinogen component has been described in the art, e.g. U.S.Pat. No. 5,716,645, and similar materials are commercially available,e.g. from sources such as Sigma-Aldrich, Enzyme Research Laboratories,Haematologic Technologies and Aniara.

The fibrinogen activator employed in the haemostatic layer(s) of thesolid dressing may be any of the substances or mixtures of substancesknown by those skilled in the art to convert fibrinogen into fibrin.Illustrative examples of suitable fibrinogen activators include, but arenot limited to, the following: thrombins, such as human thrombin orbovine thrombin, and prothrombins, such as human prothrombin orprothrombin complex concentrate (a mixture of Factors II, VII, IX andX); snake venoms, such as batroxobin, reptilase (a mixture of batroxobinand Factor XIIIa), bothrombin, calobin, fibrozyme, and enzymes isolatedfrom the venom of Bothrops jararacussu; and mixtures of any two or moreof these. See, e.g., Dascombe et al., Thromb. Haemost. 78:947-51 (1997);Hahn et al., Biochem. (Tokyo) 119:835-43 (1996); Fortova et al., J.Chromatogr. S. Biomed. Appl. 694:49-53 (1997); and Andriao-Escarso etal., Toxicon. 35: 1043-52 (1997).

Preferably, the fibrinogen activator is a thrombin. More preferably, thefibrinogen activator is a mammalian thrombin, although bird and/or fishthrombin may also be employed in appropriate circumstances. While anysuitable mammalian thrombin may be used in the solid dressing, thethrombin employed in the haemostatic layer is preferably a lyophilizedmixture of human plasma proteins which has been sufficiently purifiedand virally inactivated for the intended use of the solid dressing.Suitable thrombin is available commercially from sources such asSigma-Aldrich, Enzyme Research Laboratories, Haematologic Technologiesand Biomol International. A particularly preferred aqueous solution ofthrombin for preparing a solid dressing contains thrombin at a potencyof between 10 and 2000±50 International Units/mL, and more preferred ata potency of 25±2.5 International Units/mL. Other constituents mayinclude albumin (generally about 0.1 mg/mL) and glycine (generally about100 mM±0.1 mM). The pH of this particularly preferred aqueous solutionof thrombin is generally in the range of 6.5-7.8, and preferably7.4±0.1, although a in the range of 5.5-8.5 may be acceptable.

In addition to the haemostatic layer(s), the solid dressing mayoptionally further comprise one or more support layers. As used herein,a “support layer” refers to a material that sustains or improves thestructural integrity of the solid dressing and/or the fibrin clot formedwhen such a dressing is applied to wounded tissue.

According to certain preferred embodiments of the present invention thesupport layer comprises a backing material on the side of the dressingopposite the side to be applied to wounded tissue. Such a backingmaterial may be affixed with a physiologically-acceptable adhesive ormay be self-adhering (e.g. by having a sufficient surface staticcharge). The backing material may comprise one or more resorbablematerials or one or more non-resorbable materials or mixtures thereof.Preferably, the backing material is a single resorbable material.

Any suitable resorbable material known and available to those skilled inthe art may be employed in the present invention. For example, theresorbable material may be a proteinaceous substance, such as silk,fibrin, keratin, collagen and/or gelatin. Alternatively, the resorbablematerial may be a carbohydrate substance, such as alginates, chitin,cellulose, proteoglycans (e.g. poly-N-acetyl glucosamine), hyaluron andits derivatives, such as hyaluronic acid, glycolic acid polymers, lacticacid polymers, or glycolic acid/lactic acid co-polymers. The resorbablematerial may also comprise a mixture of proteinaceous substances or amixture of carbohydrate substances or a mixture of both proteinaceoussubstances and carbohydrate substances. Specific resorbable material(s)may be selected empirically by those skilled in the art depending uponthe intended use of the solid dressing.

According to certain preferred embodiments of the present invention, theresorbable material is a carbohydrate substance. Illustrative examplesof particularly preferred resorbable materials include, but are notlimited to, the materials sold under the trade names VICRYL™ (a glycolicacid/lactic acid copolymer) and DEXON™ (a glycolic acid polymer).

Any suitable non-resorbable material known and available to thoseskilled in the art may be employed as the backing material. Illustrativeexamples of suitable non-resorbable materials include, but are notlimited to, plastics, silicone polymers, paper and paper products,latex, gauze and the like.

According to other preferred embodiments, the support layer comprises aninternal support material. Such an internal support material ispreferably fully contained within a haemostatic layer of the soliddressing., although it may be placed between two adjacent haemostaticlayers in certain embodiments. As with the backing material, theinternal support material may be a resorbable material or anon-resorbable material, or a mixture thereof, such as a mixture of twoor more resorbable materials or a mixture of two or more non-resorbablematerials or a mixture of resorbable material(s) and non-resorbablematerial(s).

According to still other preferred embodiments, the support layer maycomprise a front support material on the wound-facing side of thedressing, i.e. the side to be applied to wounded tissue. As with thebacking material and the internal support material, the front supportmaterial may be a resorbable material or a non-resorbable material, or amixture thereof, such as a mixture of two or more resorbable materialsor a mixture of two or more non-resorbable materials or a mixture ofresorbable material(s) and non-resorbable material(s).

According to still other preferred embodiments, the solid dressingcomprises both a backing material and an internal support material inaddition to the haemostatic layer(s), i.e. the solid dressing comprisestwo support layers in addition to the haemostatic layer(s). According tostill other preferred embodiments, the solid dressing comprises both afront support material and an internal support material in addition tothe haemostatic layer(s). According to still other preferredembodiments, the solid dressing comprises a backing material, a frontsupport material and an internal support material in addition to thehaemostatic layer(s).

According to certain embodiments of the present invention, particularlywhere the solid dressing is manufactured using a mold, the soliddressings may also optionally further comprise a release layer inaddition to the haemostatic layer(s) and support layer(s). As usedherein, a “release layer” refers to a layer containing one or moreagents (“release agents”) which promote or facilitate removal of thesolid dressing from a mold in which it has been manufactured. Apreferred such agent is sucrose, but other suitable release agentsinclude gelatin, mannitol, sorbitol, hyaluron and its derivatives, suchas hyaluronic acid, and glucose. Alternatively, such one or more releaseagents may be contained in the haemostatic layer.

The various layers of the inventive dressings may be affixed to oneanother by any suitable means known and available to those skilled inthe art. For example, a physiologically-acceptable adhesive may beapplied to a backing material (when present), and the haemostaticlayer(s) subsequently affixed thereto.

In certain embodiments of the present invention, thephysiologically-acceptable adhesive has a shear strength and/orstructure such that the backing material can be separated from thefibrin clot formed by the haemostatic layer after application of thedressing to wounded tissue. In other embodiments, thephysiologically-acceptable adhesive has a shear strength and/orstructure such that the backing material cannot be separated from thefibrin clot after application of the bandage to wounded tissue.

Suitable fibrinogens and suitable fibrinogen activators for thehaemostatic layer(s) of the solid dressing may be obtained from anyappropriate source known and available to those skilled in the art,including, but not limited to, the following: from commercial vendors,such as Sigma-Aldrich and Enzyme Research Laboratories; by extractionand purification from human or mammalian plasma by any of the methodsknown and available to those skilled in the art; from supernatants orpastes derived from plasma or recombinant tissue culture, viruses,yeast, bacteria, or the like that contain a gene that expresses a humanor mammalian plasma protein which has been introduced according tostandard recombinant DNA techniques; and/or from the fluids (e.g. blood,milk, lymph, urine or the like) of transgenic mammals (e.g. goats,sheep, cows) that contain a gene which has been introduced according tostandard transgenic techniques and that expresses the desired fibrinogenand/or desired fibrinogen activator.

According to certain preferred embodiments of the present invention, thefibrinogen is a mammalian fibrinogen such as bovine fibrinogen, porcinefibrinogen, ovine fibrinogen, equine fibrinogen, caprine fibrinogen,feline fibrinogen, canine fibrinogen, murine fibrinogen or humanfibrinogen. According to other embodiments, the fibrinogen is birdfibrinogen or fish fibrinogen. According to any of these embodiments,the fibrinogen may be recombinantly produced fibrinogen or transgenicfibrinogen.

According to certain preferred embodiments of the present invention, thefibrinogen activator is a mammalian thrombin, such as bovine thrombin,porcine thrombin, ovine thrombin, equine thrombin, caprine thrombin,feline thrombin, canine thrombin, murine thrombin and human thrombin.According to other embodiments, the thrombin is bird thrombin or fishthrombin. According to any of these embodiments, the thrombin may berecombinantly produced thrombin or transgenic thrombin.

As a general proposition, the purity of the fibrinogen and/or thefibrinogen activator for use in the solid dressing will be a purityknown to one of ordinary skill in the relevant art to lead to theoptimal efficacy and stability of the protein(s). Preferably, thefibrinogen and/or the fibrinogen activator has been subjected tomultiple purification steps, such as precipitation, concentration,diafiltration and affinity chromatography (preferably immunoaffinitychromatography), to remove substances which cause fragmentation,activation and/or degradation of the fibrinogen and/or the fibrinogenactivator during manufacture, storage and/or use of the solid dressing.Illustrative examples of such substances that are preferably removed bypurification include: protein contaminants, such as inter-alpha trypsininhibitor and pre-alpha trypsin inhibitor; non-protein contaminants,such as lipids; and mixtures of protein and non-protein contaminants,such as lipoproteins.

The amount of the fibrinogen activator employed in the solid dressing ispreferably selected to optimize both the efficacy and stability thereof.As such, a suitable concentration for a particular application of thesolid dressing may be determined empirically by one skilled in therelevant art. According to certain preferred embodiments of the presentinvention, when the fibrinogen activator is human thrombin, the amountof human thrombin employed is between 2.50 Units/mg of fibrinogencomponent and 0.025 Units/mg of the fibrinogen (all values being±0.0009). Other preferred embodiments are directed to similar soliddressings wherein the amount of thrombin is between 0.250 Units/mg offibrinogen and 0.062 Units/mg of fibrinogen and solid dressings whereinthe amount of thrombin is between 0.125 Units/mg of fibrinogen and 0.080Units/mg of fibrinogen.

During use of the solid dressing, the fibrinogen and the fibrinogenactivator are preferably activated at the time the dressing is appliedto the wounded tissue by the endogenous fluids of the patient escapingfrom the hemorrhaging wound. Alternatively, in situations where fluidloss from the wounded tissue is insufficient to provide adequatehydration of the protein layers, the fibrinogen component and/or thethrombin may be activated by a suitable, physiologically-acceptableliquid, optionally containing any necessary co-factors and/or enzymes,prior to or during application of the dressing to the wounded tissue.

In some embodiments of the present invention, the haemostatic layer(s)may also contain one or more supplements, such as growth factors, drugs,polyclonal and monoclonal antibodies and other compounds. Illustrativeexamples of such supplements include, but are not limited to, thefollowing: fibrinolysis inhibitors, such as aprotonin, tranexamic acidand epsilon-amino-caproic acid; antibiotics, such as tetracycline andciprofloxacin, amoxicillin, and metronidazole; anticoagulants, such asactivated protein C, heparin, prostacyclins, prostaglandins(particularly (PGI₂), leukotrienes, antithrombin III, ADPase, andplasminogen activator; steroids, such as dexamethasone, inhibitors ofprostacyclin, prostaglandins, leukotrienes and/or kinins to inhibitinflammation; cardiovascular drugs, such as calcium channel blockers,vasodilators and vasoconstrictors; chemoattractants; local anestheticssuch as bupivacaine; and antiproliferative/antitumor drugs such as5-fluorouracil (5-FU), taxol and/or taxotere; antivirals, such asgangcyclovir, zidovudine, amantidine, vidarabine, ribaravin,trifluridine, acyclovir, dideoxyuridine and antibodies to viralcomponents or gene products; cytokines, such as alpha- or beta- orgamma-Interferon, alpha- or beta-tumor necrosis factor, andinterleukins; colony stimulating factors; erythropoietin; antifungals,such as diflucan, ketaconizole and nystatin; antiparasitic gents, suchas pentamidine; anti-inflammatory agents, such as alpha-1-anti-trypsinand alpha-1-antichymotrypsin; anesthetics, such as bupivacaine;analgesics; antiseptics; hormones; vitamins and other nutritionalsupplements; glycoproteins; fibronectin; peptides and proteins;carbohydrates (both simple and/or complex); proteoglycans;antiangiogenins; antigens; lipids or liposomes; oligonucleotides (senseand/or antisense DNA and/or RNA); and gene therapy reagents. In otherembodiments of the present invention, the backing layer and/or theinternal support layer, if present, may contain one or more supplements.According to certain preferred embodiments of the present invention, thetherapeutic supplement is present in an amount greater than itssolubility limit in fibrin.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

EXAMPLES

The ability of the dressings to seal an injured blood vessel wasdetermined by an ex vivo porcine arteriotomy (EVPA) performance test,which was first described in U.S. Pat. No. 6,762,336. The EVPAperformance test evaluates the ability of a dressing to stop fluid flowthrough a hole in a porcine artery. While the procedure described inU.S. Pat. No. 6,762,336 has been shown to be useful for evaluatinghaemostatic dressings, it failed to replicate faithfully therequirements for success in vivo. More specifically, the proceduredisclosed in U.S. Pat. No. 6,762,336 required testing at 37° C.,whereas, in the real world, wounds are typically cooler than that. Thisdecreased temperature can significantly reduce the rate of fibrinformation and its haemostatic efficacy in trauma victims. See, e.g.,Acheson et al., J. Trauma 59:865-874 (2005). The test in U.S. Pat. No.6,762,336 also failed to require a high degree of adherence of thedressing to the injured tissue. A failure mode in which fibrin forms butthe dressing fails to attach tightly to the tissue would, therefore, notbe detected by this test. Additionally, the pressure utilized in theprocedure (200 mHg) may be exceeded during therapy for some traumapatients. The overall result of this is that numerous animal tests,typically involving small animals (such as rats and rabbits), must beconducted to accurately predict dressing performance in large animal,realistic trauma studies and in the clinical environment.

In order to minimize the amount of time and the number of animal studiesrequired to develop the present invention, an improved ex vivo testingprocedure was developed. To accomplish this, the basic conditions underwhich the dressing test was conducted were changed, and the severity ofthe test parameters was increased to include testing at lowertemperatures (i.e. 29-33° C. vs. 37° C., representing the realphysiologic challenge at realistic wound temperatures (Acheson et al.,J. Trauma 59:865-874 (2005)), higher pressures (i.e. 250 mmHg vs. 200mmHg), a longer test period (3 minutes vs. 2 minutes) and larger sizedarterial injuries (U.S. Pat. No. 6,762,336 used an 18 gauge needlepuncture, whereas the revised procedure used puncture holes ranging from2.8 mm to 4 mm×6 mm).

In addition, a new test was derived to directly measure adherence of thedressing to the injured tissue. Both these tests showed greatly improvedstringency and are thus capable of surpassing the previous ex vivo testand replacing many in vivo tests for efficacy.

The following is a list of acronyms used in the Examples below:

CFB: Complete Fibrinogen Buffer (100 mM Sodium Chloride, 1.1 mM CalciumChloride, 10 Mm Tris, 10 mM Sodium Citrate, 1.5% Sucrose, Human SerumAlbumin (80 mg/g of total protein) and Tween™ 80 (animal source) 15 mg/gtotal protein)CTB: Complete Thrombin Buffer (150 mM Sodium Chloride, 40 mM CalciumChloride, 10 mM Tris and 100 mM L-Lysine with the addition of HSA at 100ug/ml)

ERL: Enzyme Research Laboratories EVPA: Ex Vivo Porcine Arteriotomy

FD: Inventive haemostatic dressing

HSA: Human Serum Albumin

HD: A “sandwich” fibrin sealant haemostatic dressing as disclosed inU.S. Pat. No. 6,762,336IFB: Incomplete Fibrinogen Buffer; CFB without HSA and Tween

PETG: Glycol-modified Polyethlylenetetrapthalate PPG: Polypropylene

PVC: Poly vinyl chlorideTRIS: trishydroxymethylaminomethane(2-amino-2-hydroxymethyl-1,3-propanediol)

Example 1

Backing material (DEXON™) was cut and placed into each PETG 2.4×2.4 cmmold. Twenty-five microliters of 2% sucrose was pipetted on top of eachof the four corners of the backing material. Once completed the moldswere placed in a −80° C. freezer for at least 60 minutes. Fibrinogen(Enzyme Research Laboratories™) was formulated in CFB. The final pH ofthe fibrinogen was 7.4±0.1. The fibrinogen concentrations were adjustedto 37.5, 31.7, 25.9, 20.16, 14.4, 8.64, and 4.3 mg/ml. When 2 ml offibrinogen was delivered into the molds, this would result in afibrinogen dose of 13, 11, 9, 7, 5, 3 or 1.5 mg/cm². Once prepared thefibrinogen was placed on ice until use. Thrombin was formulated in CTB.The final pH of the thrombin was 7.4±0.1. The concentrations of thrombinwere adjusted so that when mixed with the fibrinogen solutions asdescribed below, the combination would produce a solution that contained0.1 units/mg of Fibrinogen. Once prepared the thrombin was placed on iceuntil use. The temperature of the fibrinogen and thrombin prior todispensing was 4° C.±2° C. Molds were removed from the −80° C. freezerand placed on a copper plate that was placed on top of dry ice. A repeatpipettor was filled with fibrinogen and second repeat pipettor wasfilled with thrombin. Two ml of fibrinogen and 300 micro liters ofthrombin were dispensed simultaneously into each mold. Once the moldswere filled they were allowed to freeze and then returned to the −80° C.freezer for at least two hours. The frozen dressings were then placedinto a pre-cooled Genesis™ lyophylizer (Virtis, Gardiner, N.Y.). Thechamber was sealed and the temperature equilibrated. The chamber wasthen evacuated and the dressings lyophilized via a primary and secondarydrying cycle.

The dressings were removed from the lyophylizer, sealed in foil pouchesand stored at room temperature until testing. Subsequently, thedressings were evaluated in the EVPA, Adherence and Weight Assays.

The results are given in the following Table and depicted graphically inFIGS. 1A-1C.

Weight Weight EVPA Peel Test Adherence Held Held Group Pass/TotalAdherence Std Dev (mean) (g) Std Dev 13 mg/cm²  6/6 4.0 0.0 198.0 12.611 mg/cm²  6/6 3.8 0.4 163 48.5 9 mg/cm² 5/6 3.0 0.0 88 20.0 7 mg/cm²6/6 3.2 0.4 93 17.6 7 mg/cm² 5/6 3.0 0.0 94.7 8.2 5 mg/cm² 5/5 2.8 0.476 34.2 3 mg/cm² 5/5 2.4 0.5 48 27.4 1.5 mg/cm²   0/6 0.1 0.2 4.7 11.4

Example 2

Monolithic dressings were manufactured as follows: backing material wascut and placed into each PETG 2.4×2.4cm mold. Twenty-five microliters of2% sucrose was pipetted on top of each of the four corners of thebacking material Once completed the molds were placed in a −80° C.freezer for at least 60 minutes.

For all dressings, ERL fibrinogen lot 3114 was formulated in CFB. Thefinal pH of the fibrinogen was 7.4±0.1. The fibrinogen concentration wasadjusted to 37.5 mg/ml. Once prepared the fibrinogen was placed on iceuntil use. Thrombin was formulated in CTB. The final pH of the thrombinwas 7.4±0.1. The thrombin was adjusted to deliver 0.1 units/mg ofFibrinogen or 25 Units/ml thrombin. Once prepared the thrombin wasplaced on ice until use. The temperature of the fibrinogen and thrombinprior to dispensing was 4° C.±2° C. Molds were removed from the −80° C.freezer and placed on a copper plate that was placed on top of dry ice.A repeat pipettor was filled with fibrinogen and second repeat pipettorwas filled with thrombin. Simultaneously 2 ml of fibrinogen and 300micro liters of thrombin were dispensed into each mold. Once the moldswere filled they were returned to the −80° C. freezer for at least twohours before being placed into the freeze dryer. Dressings were thenlyophilized as described above. Once complete the dressings were storedin low moisture transmission foil bags containing 5 grams of desiccant.

Trilayer dressings were manufactured as described previously¹, using thesame materials as described above. Subsequently, the dressings wereplaced under conditions of 100% relative humidity at 37° C. for varioustimes in order to increase their relative moisture content to desiredlevels. The dressings were evaluated visually and for their handling andother physical characteristics. Following this evaluation, a sample ofeach of the dressings was tested to determine their moisture content.The remaining dressings were performance tested in the EVPA, Adherenceand Weight Held assays.

Results

The results of the assays are given in the Tables below:

TABLE 1 Performance Data of Inventive Solid Dressings Exposure TimeWeight to 100% Humidity % EVPA Peel Test Held (g) @ 37° C. Mois- #Adherence (mean ± Std. (minutes) ture Pass/Total (±Std. Dev.) Dev.) 02.5 2/2 4.0 ± 0 148 ± 28.3 1 5.8 2/2   3.5 ± 0.71 123 ± 7.1  15 16 2/2 2.5 ± .71 108 ± 14.1 45 24 2/2 4.0 ± 0 168 ± 0   60 28 2/2 4.0 ± 0 273± 7.1  225 44 2/2  2 ± 0  58 ± 14.1 1200 52 ND ND ND

TABLE 2 Performance Data for Tri-layer Dressings Exposure Time to 100%Humidity EVPA Weight @ 37° C. % # Peel Test Held (g) (minutes) MoisturePass/Total Adherence (mean) 0 3 1/1 2.0 78 15 22 1/1 2.0 78 60 33.7 0/10.5 28

TABLE 3 Integrity and Handling Characteristics of Inventive SolidDressings Exposure Time During Hydration to 100% Force Humidity Prior ToHydration Required After @ 37° C. Surface Speed of for Hydration(minutes) Appearance Curling Integrity Flexible Hydration HydrationAppearance 0 Normal No Excellent No Normal No Normal (Smooth, No (Nocracks or “skin”) flaking off) 1 Normal ″ Excellent Yes ″ ″ ″ (Smooth,No (No cracks or “skin”) flaking off) 15 Normal ″ Excellent ″ ″ ″ ″(Smooth, No (No cracks or “skin”) flaking off) 45 Normal ″ Excellent ″ ″″ ″ (Smooth, No (No cracks or “skin”) flaking off) 60 Normal SlightExcellent ″ ″ ″ ″ (Smooth, No (No cracks or “skin”) flaking off) 225Normal Yes Excellent ″ ″ ″ ″ (Smooth, No (No cracks or “skin”) flakingoff) 1200 Normal Curling Excellent ″ n/d n/d Mottled & (Smooth, No up on(No cracks or lumpy “skin”) itself flaking off)

TABLE 4 Integrity and Handling Characteristics of Trilayer DressingsExposure Time During Hydration to 100% Force Humidity Prior To HydrationRequired After @ 37° C. Surface Speed of for Hydration (minutes)Appearance Curling Integrity Flexibility Hydration Hydration Appearance0 Normal No Good; some No Normal No Normal delamination 15 Irregular NoGood; some Yes Slow No Mottled delamination 60 Skinned Yes Good; someYes Very Slow Yes Very delamination Mottled and lumpy

Conclusions:

The monolithic dressings were fully functional at very high levels ofmoisture. As much as 28% moisture was found to retain completefunctionality. When the moisture levels rose to 44%, the dressings werestill functional, however some of their activity was reduced Higherlevels of moisture may also retain some function. The originaldressings, at 2.5% moisture content, were not flexible, but had all theother desired properties including appearance, a flat surface,integrity, rapid and uncomplicated hydration and a smooth appearancepost hydration. Once the moisture content was increased to 5.8%, themonolithic dressings became flexible, while retaining theirfunctionality and desirable characteristics. They retained theirflexibility, without curling or losing their integrity or appearing toform excessive amounts of fibrin prior to hydration.

This contrasted with the tri-layer dressings, which began to lose theirdesirable characteristics upon the addition of moisture, and lost thementirely by the time moisture had increased to 33%. At no time did thesedressings become flexible.

Example 3

For dressings utilizing a backing, the backing material was cut andplaced into each PETG 2.4×2.4 cm mold. Twenty-five microliters of 2%sucrose was pipetted on top of each of the four corners of the backingmaterial. Once completed the molds were placed in a −80° C. freezer forat least 60 minutes. For dressings without backing material, PETG2.4×2.4 cm molds were placed in a −80° C. freezer for at least 60minutes.

For all dressings, ERL fibrinogen lot 3114 was formulated in CFB. Thefinal pH of the fibrinogen was 7.4±0.1. The fibrinogen concentration wasadjusted to 37.5 mg/ml. Once prepared the fibrinogen was placed on iceuntil use. Thrombin was formulated in CTB. The final pH of the thrombinwas 7.4±0.1. The thrombin was adjusted to deliver 0.1 units/mg ofFibrinogen or 25 Units/ml thrombin. Once prepared the thrombin wasplaced on ice until use. The temperature of the fibrinogen and thrombinprior to dispensing was 4° C.±2° C. Molds were removed from the −80° C.freezer and placed on a copper plate that was placed on top of dry ice.A repeat pipettor was filled with fibrinogen and second repeat pipettorwas filled with thrombin. Simultaneously 2 ml of fibrinogen and 300micro liters of thrombin were dispensed into each mold. Once the moldswere filled they were returned to the −80° C. freezer for at least twohours before being placed into the freeze dryer. Dressings were thenlyophylized as described below.

Both groups were performance tested in the EVPA assay. In addition, thegroup which had a backing was also tested in the Adherence and WeightHeld assays.

Results:

Weight Weight EVPA Peel Test Adherence Held Held Group # Pass/TotalAdherence Std Dev (mean) (g) Std Dev Backing 6/6  3.7 0.5 153 37.3 NoBacking 9/12

Conclusions:

Dressings formulated with backing material performed well, with alldressings passing the EVPA test, and high values for adherence andweight held. Dressings without backing material were not quite aseffective in the EVPA assay, however, surprisingly 75% of them passedthe EVPA test. Without the backing the other tests could not beperformed. The ability of the dressings made without a backing tosucceed in the EVPA assay indicates that these dressings would beeffective in treating arterial injuries and even more effective intreating venous and small vessel injuries.

Example 4

For all dressings, ERL fibrinogen lot 3130 was formulated in CFB. Thefinal pH of the fibrinogen was 7.4±0.1. The fibrinogen concentration wasadjusted to 37.5 mg/ml. Once prepared the fibrinogen was placed on iceuntil use. Thrombin was formulated in CTB. The final pH of the thrombinwas 7.4±0.1. The thrombin was adjusted to deliver 0.1 units/mg ofFibrinogen or 25 Units/ml thrombin. For the group with shredded VICRYL™mesh dispersed within, this support material was cut into approximately1 mm×1 mm pieces and dispersed within the thrombin solution prior tofilling the molds. Once prepared the thrombin was placed on ice untiluse. The temperature of the fibrinogen and thrombin prior to dispensingwas 4° C.±2° C. Cylindrical molds made of 10 or 3 mL polypropylenesyringes (Becton Dickinson) with the luer-lock end removed were used.The plungers were withdrawn to the 6 mL and 2 mL mark respectively. Fordressings utilizing a backing, the support material was cut and placedinto each mold and pushed down until it was adjacent to the plunger.Once prepared the molds were placed upright and surrounded by dry ice,leaving the opening exposed at the top. 1 ml of fibrinogen and 0.15 mLof thrombin (with or without backing material dispersed within) weredispensed into the 10 mL molds and 1 ml of fibrinogen and 0.15 mL ofthrombin (with or without support material dispersed within) weredispensed into the 3 mL molds, which were allowed to freeze for 5minutes. The molds were then placed into the −80° C. freezer for atleast two hours before being placed into the freeze dryer andlyophylized as described above.

Upon removal from the lyophylizer, both groups were performance testedin a modified EVPA assay. Briefly, a plastic foam form was slipped overthe artery. This covering had a hole in it that corresponded to the holein the artery and the surrounding tissue. Warm saline was added to thesurface of the dressing and the mold was immediately passed down thruthe hole in the foam to the artery surface. The plunger was thendepressed and held by hand for 3 minutes, after which the mold waswithdrawn as the plunger was depressed further. At this point the arterywas pressurized and the assay continued as before.

Results

Mold EVPA Result Maximum Support Material Size (@250 mmHg) Pressure None10 ml Pass >250 mmHg Dexon Mesh Backing 10 ml Pass ″ ″  3 ml Pass ″Shredded Dexon Mesh (Dispersed) 10 ml Pass ″ ″  3 ml Fail  150 mmHg

Conclusions:

Dressings that included no backing or a DEXON™ mesh backing performedwell, with all passing the EVPA test at 250 mmHg When the supportmaterial was dispersed throughout the composition, the dressings alsoperformed well, with the large size (10 mL mold) dressings holding thefull 250 mmHg of pressure, while the smaller held up to 150 mmHg ofpressure. This indicates that the use of a support material may beoptional, and it's location may be on the ‘back’ of the dressing, ordispersed thou the composition, as desired.

Example 5

Dressings made with a support material on the “back” (i.e. thenon-wound-facing side) of the dressing were manufactured by firstcutting the mesh support material and placing it into each PETG 10×10 cmmold. Twenty-five microliters of 2% sucrose was pipetted on top of eachof the four corners of the backing material. Once completed the moldswere placed in a −80° C. freezer for at least 60 minutes.

For dressings made with a support material on the “front” (i.e. thewound-facing side) of the dressing, these were manufactured without anysupport material in the mold. The support mesh was placed atop thedressing immediately after dispensing of the fibrinogen and thrombininto the mold (see below), and lightly pressing it into the surfaceprior to its freezing. In all other ways the manufacture of thedressings was similar as described below.

For all dressings, ERL fibrinogen lot 3114 was formulated in CFB. Thefinal pH of the fibrinogen was 7.4±0.1. The fibrinogen concentration wasadjusted to 37.5 mg/ml. Once prepared the fibrinogen was placed on iceuntil use. Thrombin was formulated in CTB. The final pH of the thrombinwas 7.4±0.1. The thrombin was adjusted to deliver 0.1 units/mg ofFibrinogen or 25 Units/ml thrombin. Once prepared the thrombin wasplaced on ice until use. The thrombin was adjusted to deliver 0.1units/mg of Fibrinogen or 25 Units/ml thrombin. Once prepared thethrombin was placed on ice until use. The temperature of the fibrinogenand thrombin prior to dispensing was 4° C.±2° C. The mold was removedfrom the −80° C. freezer and placed on an aluminum plate that was placedon top of dry ice. The aluminum plate had a 0.25 inch hole drilled inthe center and a fitting attached so that a piece of tubing could beattached to a vacuum source. The mold was centered over the hole in thealuminum plate and vacuum was turned on. The vacuum served two purposesit prevented the mold from moving and it held it flat against thealuminum plate. Thirty-five milliliters of fibrinogen and 5.25milliliters of Thrombin were placed in 50 ml test tube, inverted threetimes and poured into the mold. Once the molds were filled and thesupport material applied as described above, they were returned to the−80° C. freezer for at least two hours before being placed into thefreeze dryer. Dressings were then lyophylized as described previously.

Both groups were performance tested in the EVPA assay. In addition, thegroup which had a backing was also tested in the Adherence and WeightHeld assays.

Results:

EVPA Adher- Weight Weight # ence Adher- Held Held Support Material Pass/Test ence (mean) Std (Mesh) Orientation Total Score Std Dev (g) Dev Back(away from injury 6/6 3.5 0.5 136 49 site) Front (immediately 6/6 3.80.4 163 32 adjacent to injury site)

Conclusions:

Dressings formulated with backing material in either orientation well,with all dressings passing the EVPA test, and high values for adherenceand weight held. This indicates that the location of a support materialmay be on the ‘back’ of the dressing, or the ‘front’, of the compositionas desired.

EVPA Performance Testing

Equipment and Supplies:

-   -   In-line high pressure transducer (Ashcroft Duralife™ or        equivalent)    -   Peristaltic pump (Pharmacia Biotech™, Model P-1 or equivalent)    -   Voltmeter (Craftsman™ Professional Model 82324 or equivalent)    -   Computer equipped with software for recording pressure or        voltage information    -   Tygon™ tubing (assorted sizes) with attachments    -   Water bath (Baxter Durabath™ or equivalent), preset to 37° C.    -   Incubation chamber (VWR™, Model 1400G or equivalent), preset to        37° C.    -   Thermometer to monitor temperatures of both water bath and oven    -   Assorted forceps, hemostats, and scissors    -   10 cc. and 20 cc. syringes with an approximately 0.6 cm hole        drilled in center and smaller hole drilled through both syringe        and plunger. This hole, drilled into the end of the syringe,        will be used to keep the plunger drawn back and stationary.    -   O-rings (size 10 and 13)    -   Plastic Shields to fit the 10 cc and 20 cc syringes        (approximately 3.5 cm in length)    -   P-1000 Pipetman™ with tips    -   Sphygmomanometer with neonatal size cuff and bladder    -   Programmable Logic Controller (PLC) to control the pumps to        maintain the desired pressure profile (Optional. Manual control        may be used if desired.)

1. Materials and Chemicals

-   -   Porcine descending aortas (Pel-Freez Biologicals™, Catalog #        59402-2 or equivalent)    -   Cyanoacrylate glue (Vetbond™, 3M or equivalent)    -   18-gauge needle(s)    -   0.9% Saline, maintained at 37° C.    -   Red food coloring    -   Vascular Punch(es), 2.8 mm or other    -   Plastic Wrap

2. Artery Cleaning and Storage

-   -   1. Store arteries at −20° C. until used.    -   2. Thaw arteries at 37° C. in H₂O bath.    -   3. Clean fat and connective tissue from exterior surface of        artery.    -   4. Cut the arteries into ˜5 cm segments.    -   5. The arteries may be refrozen to −20° C. and stored until use.

3. Artery Preparation for Assay

-   -   1. Turn the artery inside-out so that the smooth, interior wall        is facing outwards.    -   2. Stretch a size 13 O-ring over a 20 cc syringe or a size 10        O-ring over a 10 cc syringe with an approximately 0.6 cm (0.25        in) hole drilled into one side.    -   3. Pull the artery onto the syringe, taking care not to tear the        artery or have a too loose fit. The artery should fit snugly to        the syringe. Slide another O-ring of the same size onto the        bottom of the syringe    -   4. Carefully pull both O-rings over the ends of the artery. The        distance between the O-rings should be at least 3.5 cm    -   5. Using the blade of some surgical scissors, gently scrape the        surface of the artery in order to roughen the surface of the        artery.    -   6. Use a 18-gauge needle to poke a hole through the artery over        the site of the hole in the syringe barrel (see note above)    -   7. The tip of the biopsy punch is inserted through the hole in        the artery. Depress the punch's plunger to make an open hole in        the artery. Repeat a couple of times to ensure that the hole is        open and free of connective tissue.    -   8. Patch holes left by collateral arteries. Generally this is        done by cutting a patch from a latex glove and gluing it over        the hole with cyanoacrylate glue. Allow the glue to cure for at        least 10 minutes.    -   9. Place the artery in the warmed, moistened container and place        in the incubation chamber. Allow the arteries to warm for at        least 30 minutes.

4. Solution and Equipment Preparation

-   -   1. Check to see that the water bath and incubation chamber are        maintained at 29-33° C.    -   2. Make sure that there is sufficient 0.9% saline in the pump's        reservoir for completion of the day's assays. Add more if        needed.    -   3. Place 0.9% saline and 0.9% saline with a few drops of red        food coloring added into containers in a water bath so that the        solutions will be warmed prior to performing the assay.    -   4. Prepare the container for warming the arteries in the        incubation chamber by lining with KimWipes™ and adding a small        amount of water to keep the arteries moist.    -   5. Check the tubing for air bubbles. If bubbles exist, turn on        the pump and allow the 0.9% saline to flow until all bubbles are        removed.

5. Application of the Dressing

-   -   1. Open the haemostatic dressing pouch and remove haemostatic        dressing    -   2. Place the haemostatic dressing, mesh backing side UP, over        the hole in the artery    -   3. Slowly wet the haemostatic dressing with an amount of saline        appropriate for the article being tested

NOTE: A standard (13-15 mg/cm² of fibrinogen) 2.4×2.4 cm haemostaticdressing should be wet with 800 μl of saline or other blood substitute.The amount of saline used can be adjusted depending on the requirementsof the particular experiment being performed; however, any changesshould be noted on the data collection forms.

NOTE: Wet the haemostatic dressing drop wise with 0.9% saline warmed to29-33° C. or other blood substitute, taking care to keep the saline fromrunning off the edges. Any obvious differences in wettingcharacteristics from the positive control should be noted on datacollection forms.

-   -   4. Place the shield gently onto the haemostatic dressing, taking        care that it lies flat between the O-rings. Press lightly to        secure in place    -   5. Wrap the artery and haemostatic dressing with plastic wrap    -   6. Wrap with blood pressure cuff, taking care that the bladder        is adjacent to the haemostatic dressing.    -   7. Pump up the bladder to 100-120 mmHg, and monitor the pressure        and pump again if it falls below 100 mmHg. Maintain pressure for        5 minutes.

NOTE: Time and pressure can be altered according to the requirements ofthe experiment; changes from the standard conditions should be noted onthe data collection forms.

-   -   8. After polymerization, carefully unwrap the artery and note        the condition of the haemostatic dressing. Any variation from        the positive control should be noted on the data collection        form.

EXCLUSION CRITERION: The mesh backing must remain over the hole in theartery. If it has shifted during the polymerization and does notcompletely cover the hole the haemostatic dressing must be excluded.

Testing Procedure

6. Diagram of Testing Equipment Set-Up

The set-up of the testing equipment is shown in FIG. 2. Some additional,unshown components may be utilized to read out (pressure gauge) orcontrol the pressure within the system.

7. Equipment and Artery Assembly

Fill the artery and syringe with red 0.9% saline warmed to 37° C.,taking care to minimize the amount of air bubbles within the syringe &artery. Filling the artery with the opening uppermost can assist withthis. Attach the artery and syringe to the testing apparatus, makingsure that there are as few air bubbles in the tubing as possible. Theperistaltic pump should be calibrated so that it delivers approximately3 ml/min. If available, the PLC should be operated according to apre-determined range of pressures and hold times as appropriate for thearticle being tested. If under manual control, the pressure/time profileto be followed is attained by manually turning the pump on and off whilereferencing the system pressure as read out by one or morepressure-reading components of the system. Following the conclusion oftesting, the haemostatic dressing is subjectively assessed with regardto adhesion to the artery and formation of a plug in the artery hole.Any variations from the positive control should be noted on the datacollection form.

Success Criteria

Haemostatic dressings that are able to withstand pressures for 3 minutesare considered to have passed the assay. When a haemostatic dressing hassuccessfully passed the assay the data collection should be stoppedimmediately so that the natural decrease in pressure that occurs in theartery once the test is ended isn't included on the graphs. Should theoperator fail to stop data collection, these points can be deleted fromthe data file to avoid confusing the natural pressure decay that occurspost-test with an actual dressing failure. The entire testing periodfrom application of the haemostatic dressing to completion must fallwithin pre-established criteria. The maximum pressure reached should berecorded on the data collection form.

NOTE: Typical challenge is 250 mmHg for three minutes in one step, butthat may be altered based on the article being tested. Changes from thestandard procedure should be noted on the data collection forms.

Failure Criteria

Haemostatic dressings that start leaking saline at any point duringtesting are considered to have failed the assay.

NOTE: Build failures that are caused by artery swelling can be ignoredand the test continued or re-started (as long as the total testing timedoesn't fall beyond the established limit)

When leakage does occur, the pressure should be allowed to fall ˜20 mmHgbefore data collection is stopped so that the failure is easily observedon the graphs. The pressures at which leakage occurred should berecorded on the data collection form. Should the data collection stop inthe middle of the experiment due to equipment failure the data can becollected by hand at 5 second intervals until the end of the test orhaemostatic dressing failure, whichever happens first. The data pointsshould be recorded on the back of the data collection form, clearlylabeled, and entered by hand into the data tables.

Exclusion Criteria

If the total testing period exceeds the maximum allowed for thatprocedure, regardless of cause, results must be excluded. If there areleaks from collaterals that can't be fixed either by patching or fingerpressure the results must be excluded. If the test fails because ofleaks at the O-rings, the results must be excluded. If the mesh backingdoes not completely cover the hole in the artery, the results must beexcluded.

Adherence Performance Testing

8. Equipment and Supplies

Hemostat(s), Porcine artery and haemostatic dressing (usually aftercompletion of the EVPA Assay although it does not need to be performedto do the Adherence Assay).

9. Preparation of the Artery+Dressing

After application of the dressing without completion of the EVPA Assay,the dressing is ready for the Adherence Assay and Weight Limit Test (ifapplicable). After application of the dressing and subsequent EVPAAnalysis, the artery and syringe system is then disconnected slowly fromthe pump so that solution does not spray everywhere. The warmed, redsaline solution from the EVPA Assay remains in the syringe until theAdherence Assay and Weight Limit Test (if applicable) is completed.

Performance of the Adherence Assay

-   -   1. After preparation of the artery and dressing (with or without        EVPA analysis), gently lift the corner of the mesh and attach a        hemostat of known mass to the corner.

NOTE: If the FD developed a channel leak during the performance of theEVPA Assay, test the adherence on the opposite of the haemostaticdressing to obtain a more accurate assessment of the overall adherence.

-   -   2. Gently let go of the hemostat, taking care not to allow the        hemostat to drop or twist. Turn the syringe so that the hemostat        is near the top and allow the hemostat to peel back the dressing        as far as the dressing will permit. This usually occurs within        10 seconds. After the hemostat has stopped peeling back the        dressing, rate the adherence of the bandage according to the        following scale:

Dressing Performance Score Amount of Adherence 4  90+% 3 75-90% 2 50-75%1  ~50% 0.5 Only the plug holds the hemostat 0 No adherence

Exclusion Criteria

The mesh backing must remain over the hole in the artery. If it hasshifted during the polymerization and does not completely cover the holethe haemostatic dressing must be excluded.

Success Criteria

Dressings that are given an adherence score of 3 are considered to havepassed the assay.

Failure Criteria

If a dressing does not adhere to the artery after application and/orprior to performing the EVPA assay, it is given a score of 0 and failsthe adherence test. If a dressing receives a score ≦2, the dressing isconsidered to have failed the Adherence Assay.

Weight Held Performance Assay

After the initial scoring of the “Adherence Test”, weights may then beadded to the hemostat in an incremental manner until the mesh backing ispulled entirely off of the artery. The maximum weight that the dressingholds is then recorded as a measure of the amount of weight the dressingcould hold attached to the artery.

Moisture Assay

Moisture determinations were carried out using a Brinkman MetrohmMoisture Analyzer System. The system contains the following individualcomponents, 774 Oven Sample Processor, 774SC Controller, 836 Titrando, 5ml and 50 ml 800 Dosino Units and a 801 Stirrer. The system wasconnected to a computer using the Brinkman Tiamo software for datacollection, analysis and storage. The moisture system is set-up and runaccording to the manufactures recommendations and specifications tomeasure the moisture content of lyophilized samples using the KarlFischer method.

All components were turned on and allowed to reach operating temperatureprior to use. Lactose and water were run as standards and to calibratethe instrument. Once the machine was successfully calibrated, sampleswere prepared as follows. Dressing pieces weighing at least 30 mg wereplaced into vials and capped. The vials were placed in the 774 OvenSample Processor in numerical order, and one empty capped vial is placedin the conditioning space. The machine was then run to determine themoisture content (residual moisture) in the controls and samples.

1. A solid dressing for treating wounded tissue in a mammal comprisingat least one haemostatic layer consisting essentially of fibrinogen anda fibrinogen activator, wherein said fibrinogen is present in an amountbetween 3.0 mg/cm² of the wound facing surface of said dressing and 13.0mg/cm² of the wound facing surface of said dressing.
 2. The soliddressing of claim 1, further comprising at least one support layer. 3.The solid dressing of claim 2, wherein said support layer comprises abacking material.
 4. The solid dressing of claim 2, wherein said supportlayer comprises an internal support material.
 5. The solid dressing ofclaim 2, wherein said support layer comprises a resorbable material. 6.The solid dressing of claim 2, wherein said support layer comprises anon-resorbable material.
 7. The solid dressing of claim 6, wherein saidnon-resorbable material is selected from the group consisting of silconepolymers, gauze and latexes.
 8. The solid dressing of claim 3, furthercomprising at least physiologically acceptable adhesive between saidhaemostatic layer and said backing layer.
 9. The solid dressing of claim5, wherein said resorbable material is selected from the groupconsisting of proteinaceous materials and carbohydrate substances. 10.The solid dressing of claim 9, wherein said proteinaceous material is atleast one substance selected from the group consisting of keratin, silk,fibrin, collagen and gelatin.
 11. The solid dressing of claim 9, whereinsaid carbohydrate substance is selected from the group consisting ofalginic acid and salts thereof, chitin, chitosan, cellulose, n-acetylglucosamine, proteoglycans, glycolic acid polymers, lactic acidpolymers, glycolic acid/lactic acid co-polymers and mixtures of two ormore thereof.
 12. The solid dressing of claim 1, wherein saidhaemostatic layer also contains a fibrin cross-linker and/or a source ofcalcium ions.
 13. The solid dressing of claim 1, wherein saidhaemostatic layer also contains one or more of the following: at leastone filler, at least one solubilizing agent, at least one foaming agentand at least one release agent.
 14. The solid dressing of claim 13,wherein said filler is selected from the group consisting of sucrose,lactose, maltose, keratin, silk, fibrin, collagen, gelatin, albumin,polysorbate, chitin, chitosan, alginic acid and salts thereof,cellulose, proteoglycans, glycolic acid polymers, lactic acid polymers,glycolic acid/lactic acid co-polymers, and mixtures of two or morethereof.
 15. The solid dressing of claim 13, wherein said solubilizingagent is selected from the group consisting of sucrose, lactose,maltose, dextrose, mannose, trehalose, mannitol, sorbitol, albumin,sorbate, polysorbate, and mixtures of two or more thereof.
 16. The soliddressing of claim 13, wherein said release agent is selected from thegroup consisting of gelatin, mannitol, sorbitol, polysorbate, sorbitan,lactose, maltose, trehalose, sorbate, glucose and mixtures of two ormore thereof.
 17. The solid dressing of claim 13, wherein said foamingagent is selected from the group consisting of mixtures of sodiumbicarbonate/citric acid, sodium bicarbonate/acetic acid, calciumcarbonate/citric acid and calcium carbonate/acetic acid.
 18. The soliddressing of claim 1, wherein said haemostatic layer also contains atleast one therapeutic supplement selected from the group consisting ofantibiotics, anticoagulants, steroids, cardiovascular drugs, growthfactors, antibodies (poly and mono), chemoattractants, anesthetics,antiproliferatives/antitumor agents, antivirals, cytokines, colonystimulating factors, antifungals, antiparasitics, antiinflammatories,antiseptics, hormones, vitamins, glycoproteins, fibronectin, peptides,proteins, carbohydrates, proteoglycans, antiangiogenins, antigens,nucleotides, lipids, liposomes, fibrinolysis inhibitors and gene therapyreagents.
 19. The solid dressing of claim 1, wherein said haemostaticlayer is made from a single aqueous solution containing a mixture ofsaid fibrinogen and said fibrinogen activator.
 20. The solid dressing ofclaim 1, wherein said haemostatic layer is cast as a single piece. 21.The solid dressing of claim 1, wherein said haemostatic layer issubstantially homogeneous throughout.
 22. The solid dressing of claim 1,wherein said haemostatic layer is composed of a plurality of particles,each of said particles consisting essentially of fibrinogen andthrombin.
 23. The solid dressing of claim 22, wherein said haemostaticlayer further contains at least one binding agent in an amount effectiveto improve the adherence of said particles to one another.
 24. The soliddressing of claim 23, wherein said binding agent is selected from thegroup consisting of sucrose, mannitol, sorbitol, gelatin, maltose,povidone, chitosan and carboxymethylcellulose.
 25. The solid dressing ofclaim 1, wherein said haemostatic layer is a monolith.
 26. The soliddressing of claim 1, wherein said haemostatic layer has beenlyophilized.
 27. The solid dressing of claim 1, wherein said haemostaticlayer has moisture content of at least 6%.
 28. The solid dressing ofclaim 1, wherein said haemostatic layer has moisture content of lessthan 6%.
 29. The solid dressing of claim 1, wherein said fibrinogen is amammalian fibrinogen.
 30. The solid dressing of claim 29, wherein saidmammalian fibrinogen is selected from the group consisting of bovinefibrinogen, porcine fibrinogen, ovine fibrinogen, equine fibrinogen,caprine fibrinogen, feline fibrinogen, canine fibrinogen, murinefibrinogen and human fibrinogen.
 31. The solid dressing of claim 1,wherein said fibrinogen is selected from the group consisting of birdfibrinogen and fish fibrinogen.
 32. The solid dressing of claim 29 or31, wherein said fibrinogen is selected from the group consisting ofrecombinantly produced fibrinogen and transgenic fibrinogen.
 33. Thesolid dressing of claim 1, wherein said fibrinogen activator is selectedfrom the group consisting of thrombins, prothrombins, snake venoms, andmixtures of any two or more thereof.
 34. The solid dressing of claim 33,wherein said thrombin is mammalian thrombin.
 35. The solid dressing ofclaim 34, wherein said mammalian thrombin is selected from the groupconsisting of bovine thrombin, porcine thrombin, ovine thrombin, equinethrombin, caprine thrombin, feline thrombin, canine thrombin, murinethrombin and human thrombin.
 36. The solid dressing of claim 34, whereinsaid thrombin is selected from the group consisting of bird thrombin andfish thrombin.
 37. The solid dressing of claim 34 or 36, wherein saidthrombin is selected from the group consisting of recombinantly producedthrombin and transgenic thrombin.
 38. The solid dressing of claim 34,wherein said thrombin is present in an amount between 2.50 Units/mg offibrinogen component and 0.025 Units/mg of the fibrinogen.
 39. The soliddressing of claim 18, wherein said therapeutic supplement is present inan amount equal to or greater than its solubility limit in fibrin.
 40. Amethod of treating wounded tissue in a mammal, comprising placing asolid dressing of claim 1 to said wounded tissue and applying sufficientpressure to said dressing for a sufficient time for enough fibrin toform to reduce the loss of blood and/or other fluid from said woundedtissue.